Rbp3 in Geomyid rodents : reduced rate of molecular evolution or evidence for selection?

TEXAS TECH UNIVERSITY

Natural Science Research Laboratory

Occasional Papers

Museum of Texas Tech University

Number 296 25 August 2010

RbpI in Geomyid Rodents: Reduced Rate of Molecular Evolution or

Evidence for Selection?

Robert D. Bradley, Cody W. Thompson, and Ryan R. Chambers

Abstract

DNA sequences from the interphotoreceptor retinoid binding protein gene ( Rbp3 ) in pocket
gophers ( Geomys ) display an unusually slow rate of molecular evolution relative to other species
of rodents. Rates of molecular evolution were examined in pocket gophers and other members
of the rodent superfamily Geomyoidea to determine if this phenomenon was restricted to pocket
gophers. DNA sequences from the Rbp3, mitochondrial 12S ribosomal RNA(12S rRNA), and
mitochondrial cytochrome-6 (Cytb) genes were compared within members of Geomys, among
members of the Geomyidae, and among members of the Geomyoidea to ascertain rates of
molecular evolution for the three genes among the various taxa. A variety of analyses (genetic
distance, Tajima’s relative rate test, Tajima’s neutrality test, coalescence theory, and Hudson,
Kreitman, and Aguade test) indicated that DNA sequences affiliated with Rbp3 in species of
Geomys were evolving at a rate slower than were sequences of members of the Heteromyidae.
In addition, there was weak evidence suggesting that the Rbp3 gene in other pocket gopher
genera ( Cratogeomys, Orthogeomys, Pappogeomys, and Thomomys ) evolved more slowly than
in members of the Heteromyidae.

Key words: geomyoid rodents, Geomys, interphotoreceptor retinoid binding protein,
molecular evolution, pocket gophers, Rbp3

Introduction

Pocket gophers of the genus Geomys are fossorial
rodents distributed throughout the central plains and
southeastern regions of the United States and coastal
regions of northeastern Mexico (Russell 1968; Hall
1981; Baker et al. 2003; Patton 2005). Distributions
of pocket gophers are affected by availability of suit¬
able soil types (Davis 1940; Baker et al. 2003), and as
a result, populations generally contain few individuals

and are isolated from other conspecific populations.
Pocket gophers also are highly territorial leading to a
solitary lifestyle with limited vagility (Williams and
Baker 1976; Smolen et al. 1980) and non-overlapping
home ranges. In addition, past glacial events in the
central plains region are thought to have had a major
impact on speciation and distributions of members of
Geomys (Russell 1968; Hart 1978). Studies of genetic

2

Occasional Papers, Museum of Texas Tech University

evolution indicate that pocket gophers (probably as a
consequence of the above factors) have small effective
population sizes and possess low levels of intrapopu-
lational and intraspecific variation; however, variation
among populations and species is high and overall
levels of heterozygosity is low (Selander et al. 1975;
Penny and Zimmerman 1976; Avise et al. 1979; Zim¬
merman and Gay den 1981; Ruedi et al. 1997).

Recent studies pertaining to systematic relation¬
ships among species in Geomys have produced DNA
sequence data for two mitochondrial genes (12S ribo-
somal RNA- 12S rRNA, Jolley et al. 2000; cytochrome-
b - Cytb, Sudman et al. 2006) and one nuclear gene
(interphotoreceptor retinoid binding protein - Rbp3,
Chambers et al. 2009). Although the goals of these
studies were to reconstruct phylogenetic relationships
among taxa, Chambers et al. (2009) noted unusually
low levels of genetic divergence among species for
Rbp3 relative to the other two genes. Specifically,
Chambers et al. (2009) reported an average between
species genetic divergence of 0.60% (0.08%-1.5%) for
the Rbp3 gene, whereas similar comparisons among
the same taxa yielded divergence values of 3.67%
(0.6%-8.1%) for 12S rRNA and 13.8% (8.1%-21.0%)
for Cytb. Although it is well known that nuclear genes
evolve at slower rates than do mitochondrial genes, the
low level of genetic divergence associated with Rbp3

was unexpected given the higher levels of genetic
divergence reported for other rodent taxa (Stanhope et
al. 1996; Weksler 2003).

The goals of this study were to determine: 1)
whether the low rate of molecular evolution in Rbp3,
as reported by Chambers et al. (2009), is restricted to
Geomys - or is it typical for other genera of pocket go¬
phers, and 2) if the rate of molecular evolution in Rbp3
is a product of the following scenarios: a) population
dynamics, b) age of the geomyid lineage, c) reduction
of vision as a product of a fossorial lifestyle, or d) selec¬
tive forces by examining rates of molecular evolution
for genes unrelated to Rbp3 (12S rRNA and Cytb). To
examine these goals, DNA sequences were obtained for
Rbp3, 12S rRNA, and Cytb in other genera of pocket
gophers ( Cratogeomys, Orthogeomys, Pappogeomys,
and Thomomys,) and five genera of the rodent family
Heteromyidae ( Chaetodipus, Dipodomys, Heteromys,
Liomys , and Perognathus). The Heteromyidae (kan¬
garoo rats and pocket mice) is sister to the Geomyidae
and together the two families comprise the superfamily
Geomyoidea. In general, the Heteromyidae possess
larger population sizes and presumably a greater de¬
pendence on vision, and therefore offer an opportunity
to examine the four scenarios presented above in taxa
that have different demographic and natural history
traits than the Geomyidae.

Methods

Taxonomic sampling. —DNA sequences for Rbp3 ,
12S rRNA, and Cytb were either generated in this study
or obtained from GenBank for 29 individuals from the
Geomyidae: Geomys (21 individuals representing 12
species), Cratogeomys (2 individuals representing 2
species), Orthogeomys (1 individual), Pappogeomys (2
individuals from 1 species), and Thomomys (3 individu¬
als representing 3 species) and 13 individuals from the
Heteromyidae: Chaetodipus (3 individuals representing
3 species), Dipodomys (4 individuals representing 4
species), Heteromys (1 individual), Liomys (2 indi¬
viduals representing 2 species), and Perognathus (4
individuals representing 3 species). Three individuals
representing Castor canadensis were used for outgroup
comparisons. GenBank accession numbers and mu¬
seum voucher numbers are provided in Table 1.

PCR and sequencing methods. —Twenty unre¬
ported Rbp3 sequences were obtained in this study.
Genomic DNA was isolated from approximately 0.1
g of frozen liver or muscle tissue using the Puregene
DNA isolation kit (Gentra, Minneapolis, Minnesota).
Approximately 1,230 bp near the 5’ end of exon 1 of
the single-copy Rbp3 gene was amplified by the poly¬
merase chain reaction (PCR, Saiki et al. 1988) using
primers A, B, D, D2, E2, F, 125F, G, and I (Stanhope et
al. 1992; Jansa and Voss 2000; DeBry and Sagel 2001;
Weksler 2003; Chambers et al. 2009). Thermal profiles
were adapted from those of Jansa and Weksler (2004):
initial denaturation at 95°C for 10 min, 35 cycles of
denaturation at 95°C for 25 sec, annealing at 58°C for
20 sec, and extension at 72°C for 60 sec, and a final
extension at 72°C for 10 min.

Bradley et al.— Rbp3 in Geomyid Rodents

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PCR products were purified using the Exosap-II
PCR purification kit (USB Corp., Cleveland, Ohio).
Amplified gene products were sequenced on an ABI
3100-Avant using ABI Prism Big Dye v3.1 terminator
technology (Applied Biosystems, Foster City, Califor¬
nia). Primers used to cycle sequence Rbp3 included
B, D, E2, F, 125F, Geo395R, Geo609F, Geo958R,
Geol405R, and 1000F, (Stanhope et al. 1992; Jansa
and Voss 2000; DeBry and Sagel 2001; Weksler 2003;
Chambers et al. 2009). Primers beginning with “Geo”
were modified from Stanhope et al. (1992) by altering
nucleotides so they matched sequences of Geomys more
specifically. Cycle sequencing reactions were purified
using isopropanol cleanup protocols. Sequences were
assembled and proofed using Sequencher 4.9 software
(Gene Codes, Ann Arbor, Michigan) and chromato¬
grams were examined to verify all base changes and
to inspect sequences for heterozygous sites, which
were coded following the International Union of
Biochemistry (IUB) polymorphic code. MEGA 4.1
software (Kumar et al. 2007) was used to align and
inspect sequences for the presence of stop codons and
pseudogenes.

Data Analyses .—To examine rates of molecular
evolution in the three genes examined in this study
(Rbp3 - 1,230 bp, 12S rRNA- 870 bp, and Cytb -1,140
bp), five methods were implemented for data analysis.
First, neighbor-joining trees (Saitou andNei 1987) were
generated independently using DNA sequences from
each of the three genes so that taxonomic relationships
and corresponding branch lengths (indicating rates of
molecular evolution) could be compared among genes.
The neighbor joining analyses used uncorrected-P
genetic distances obtained using the MEGA 4.1 soft¬
ware (Kumar et al. 2007) for each of three respective
genes. The uncorrected-P distance was selected to
avoid interjecting “rules of molecular evolution” on
the DNA sequences as incorporated by the various
substitution models commonly used in calculating
genetic distances. This choice was crucial so that rates
of molecular evolution could be compared as evenly
as possible among nuclear and mitochondrial genes.
Average uncorrected-P distances were estimated for
individuals within each genus and between genera and
used to estimate levels of genetic divergence between
various taxonomic groups.

6

Occasional Papers, Museum of Texas Tech University

Second, Tajima’s relative rate test (Tajima
1993) using MEGA 4.1 software (Kumar et al. 2007)
was used to ascertain if rates of molecular evolution
differed significantly among taxa and among genes.
Specifically, this test was implemented to determine
if Rbp3 sequences in Geomys, and geomyids in gen¬
eral, were evolving at rates different (i.e., evidence
for rate heterogeneity) than those of heteromyids
relative to DNA sequences from 12S rRNA and Cytb.
Pairwise comparisons of DNA sequences from each
of the three genes were made between species within
Geomys , between species of Geomys and other pocket
gophers, and between geomyids and members of the
Heteromyidae.

Third, Tajima’s neutrality test (D-statistic, Tajima
1989) using MEGA 4.1 software (Kumar et al. 2007)
was implemented to determine if DNA sequences were
evolving under a neutral model of evolution (Kimura
1983) or under non-random models normally associated
with selective forces (directional selection, balancing
selection, demographic expansion or contraction, ge¬
netic hitchhiking, etc.). Specifically, the neutral model
of evolution would be operative, and would remain a
viable hypothesis, if rates of molecular evolution at
the three loci were not significantly different among
Geomys and other members of the Geomyoidea.

Fourth, the Hudson, Kreitman, and Aguade test
(HKAtest, Hudson et al. 1987) was used to determine
if the Rbp3 was behaving in a neutral fashion relative
to 12S rRNA and Cytb. The HKAtest estimates theta
(0) from the following equation, 0 = 4N e p, where N e is
the effective population size and p is the mutation rate.
Theta is estimated for each locus based on comparing
the intrapopulational genetic variability for one taxon
with the interpopulational genetic variability between
that taxon and a second. The DnaSP software pro¬
gram (version 5.10.01, Librado and Rozas 2009) was
used to estimate theta values at each locus (Rpb3,\2S
rRNA, and Cytb) for six genera of geomyoid rodents
(Chaetodipus, Cratogeomys, Dipodomys, Geomys,
Perognathus, and Thomomys) and one outgroup taxon
(Castor). A chi-square test (P < 0.05) was used to iden¬
tify significant differences among pairwise comparisons
of the three loci, with one locus representing observed
values and the second locus representing expected

values. Significantly different 0 values indicated a
deviation from neutrality (i.e. selection), with positive
selection inferred if ps for each locus were equal and
the N e was unequal and purifying selection inferred if
ps for each locus were unequal and the N e was equal.
In other words, under a model of neutrality, (Kimura
1983; Hudson et al. 1987) all loci are expected to pos¬
sess equal ps if all taxa have the same the N e ; however if
taxa have unequal N es , then positive selection acts upon
individual loci producing an excess of polymorphisms
between species, conversely, if taxa possess unequal
ps, then purifying selection generates an excess of
polymorphisms within a species.

Fifth, coalescence theory was used to estimate
the time of divergence from a hypothetical common
ancestor based on DNA sequences from the three genes.
If Rbp3 sequences coalesce at times similar to those
obtained for 12S rRNA and Cytb, then the hypothesis
of a slower rate of Rbp3 evolution in Geomys could
be rejected. The software program BEAST vl.5.3
(Drummond and Rambaut 2007) was used to analyze
the coalescence process among each gene. All taxa
were grouped into all possible taxon sets (e.g., Castori-
morpha, Geomyoidea, Geomyidae, Geomyini, Geomys,
etc.). Two fossil calibrations of ancestral taxa (Casto-
rimorpha - 54.4 MYA, McKenna 1960; Geomyoidea
- 45.45 MYA, Walsh 1991) were used as priors on the
tree. A normal distribution was used for all point fossil
calibrations with standard deviations based on dates
from the International Commission on Stratigraphy
(Gradstein et al. 2004; Ogg et al. 2008). A relaxed,
uncorrelated lognormal clock was used with a GTR +
I + G model of substitution based on MrModeltest 2.3
(Nylander 2004) and the Akaike information criterion
(Nylander 2004) for each gene. In addition, a Yule
species prior was used to date nodes within each gene
tree. Each dataset was analyzed twice for 10,000,000
generations (with a 10% burn-in) to obtain an appropri¬
ate effective sample size. The log files were combined
using LogCombiner vl.5.2 (Drummond and Rambaut
2007) and analyzed for convergence in Tracer vl.4.1
(Rambaut and Drummond 2007). A one-way analysis
of variance (ANOVA, P < 0.05) was used to compare
the mean rates of substitution to determine whether
genes were evolving at different rates.

Bradley et al.— Rbp3 in Geomyid Rodents

7

Results

Taxonomic relationships and genetic divergence .—
Genetic divergence values, based on uncorrected-P
distances, were estimated for individuals within each
genus and between genera for the three respective genes
(Table 2). Within genera values ranged from 0.66% for
individuals within Geomys to 5.98% within Liomys for
Rbp3, from 2.38% for individuals within Perognathus
to 12.02% in Chaetodipus for 12S rRNA, and from
11.93% for individuals within Geomys to 18.67% in
Cratogeomys for Cytb. In addition, these values were
used to construct a neighbor-joining tree for each of
the three genes (Fig. 1). Topologies recovered in the
three analyses were similar, although placement of
some heteromyid genera differed depending on which
gene was analyzed. However, branch lengths, reflect¬
ing the number of substitutions per site, were different
between genes and between taxa in each tree. For
example, in all analyses, branch lengths for individual
species of Geomys were substantially shorter than for
other taxa.

Rate heterogeneity. —Tajima’s relative rate test
(Tajima 1993) depicted specific taxa that exhibited dif¬
ferential rates of molecular evolution relative to other
members of the Geomyoidea based on comparisons
within each of the three genes (Table 3). In most intra¬
generic comparisons, the 12sRNAgene accounted for a
greater number of significantly different rates (P < 0.05)
than the other two genes. However, in comparisons
involving members of Geomys versus heteromyids and
geomyids versus heteromyids, Rbp3 depicted a greater
number of significantly different rates (Table 3).

Neutral model of molecular evolution .—DNA
sequences obtained from the three genes were tested
independently for departure from the model of neutral¬
ity using Tajima’s neutrality test (Tajima 1989) and the
HKA test (Hudson et al. 1987). Tajima’s neutrality
test provided evidence of positive selection or a previ¬
ous history of having been subjected to a population
bottleneck in four instances (Table 4). Two cases

Table 2. Average genetic distances (uncorrected-P distances) were estimated for each of the three genes examined
in this study. Values were estimated by averaging genetic distances for comparisons of selected taxa. Those with a
single sequence prohibited the calculation of an average distance and are indicated by N/A. Abbreviations are as fol¬
lows: interphotoreceptor retinoid binding protein gene (RbpJf mitochondrial 12S ribosomal RNA (12S rRNA), and
mitochondrial cytochrome -b (Cytb).

Taxon

Rbp3

12S rRNA

Cytb

Within Geomys

0.00661

0.03568

0.11926

Within Cratogeomys

0.1771

N/A

0.18670

Within Pappogeomys

N/A

N/A

N/A

Within Thomomys

0.02520

0.07732

0.17970

Within Chaetodipus

0.01749

0.12022

0.15510

Within Dipodomys

0.01981

0.11107

0.15341

Within Liomys

0.05976

0.05472

0.15263

Within Perognathus

0.01439

0.02375

0.16910

Within Geomyidae

0.02193

0.06869

0.15867

Within Heteromyidae

0.08326

0.16319

0.21493

Within Geomyoidea

0.07862

0.14693

0.20546

8

Occasional Papers, Museum of Texas Tech University

Rbp3

Castor (1/3)

12S rRNA

r"> Cratogeomys (2/2)
t-3- Orthogeomys (1/1)
Pappogeomys (1/2)

\

Geomys (12/21)

L-U

homomys (3/3)
Dipodomys (4/4)

Chaetodipus (3/3)

Perognathus (3/3)

Heteromys (1/1)
-> Liomys (2/2)

r 1 } Castor {M3)

■3- Cratogeomys (1/1)
Pappogeomys (1/2)

r \

^ Geomys (12/21)

_> Thomomys (2/2)

Dipodomys (4/4)

" 3 - Chaetodipus (2/2)

£■> Perognathus (2/2)

3- Heteromys (1/1)

Cyfb

■f} Castor (1/3)

£

_3" Cratogeomys (2/2)

—3" Orthogeomys (1/1)
■c> Pappogeomys {M2)

tE

■ J- Thomomys (3/3)

£

hE

7^- Dipodomys (4/4)
-J- Chaetodipus (3/3)

Liomys (2/2)

Perognathus (3/3)

■-3“ Heteromys (1/1)

M > L/omys (2/2)

0.05 substitutions/site

0.05 substitutions/site

0.05 substitutions/site

Figure 1. Neighbor joining trees obtained from uncorrected-P genetic distances estimated from DNA sequences
obtained from the Rbp3 , 12S rRNA, and Cytb genes. Only genera are labeled and numbers in parentheses following
each genus represent: number of species included per genus (left of slash), and number of DNA sequences included
per genus (right of slash).

Bradley et al.— Rbp3 in Geomyid Rodents

9

Table 3. Number of significant differences (V < 0.05) in pair-wise comparisons based on Tajima's relative rate test
(Tajima 1993). Numbers to left of the slash represent the number ofsignificant comparisons and numbers to right of the
slash indicate the number of comparisons attempted. Abbreviations are as follows: interphotoreceptor retinoid binding
protein gene (RbpJ,), mitochondrial 12S ribosomal RNA (12SrRNA), and mitochondrial cytochrome-b (Cyt b).

Taxon

Rbp3

12S rRNA

Cytb

Within Geomys

5/66

9/66

0/66

Within Cratogeomys

0/1

0/0

0/1

Within Pappogeomys

0/0

0/0

0/0

Within Thomomys

0/3

0/1

0/3

Within Chaetodipus

0/3

0/1

0/3

Within Dipodomys

0/6

3/6

1/6

Within Liomys

1/1

0/1

0/1

Within Perognathus

0/3

0/1

1/3

Geomys to Other Geomyids

0/84

1/48

3/84

Within Geomyids

4/171

10/120

7/171

Within Heteromyids

3/78

10/55

7/78

Geomys to Heteromyids

81/156

20/132

15/156

Geomyids to Heteromyids

124/247

27/176

25/247

Table 4. Results from Tajima’s neutrality test (Tajima 1993) for each of the three genes examined. The outcome of Ta¬
jima ’s neutrality test is based on Tajima’s D statistic. Taxa with sample sizes of <3 gave inconclusive results and were
not included. Abbreviations are as follows: interphotoreceptor retinoid binding protein gene fRbpi^), mitochondrial
12S ribosomal RNA (12S rRNA), mitochondrial cytochrome-b (Cytb), population bottleneck (PB), positive selection
(PS), and balancing selection (BS).

Taxon

Rbp3

12S rRNA

Cytb

Within Geomys

PB or PS (90% Cl)

PB or PS (<90% Cl)

BS

Within Dipodomys

BS

BS

BS

Within Geomyids

PB or PS (<90% Cl)

PB or PS (<90% Cl)

BS

Within Heteromyids

BS

BS

BS

Within Castorimorphs

BS

BS

BS

10

Occasional Papers, Museum of Texas Tech University

involved comparisons of taxa within Geomys ( Rbp3
and 12S rRNA) and two cases involved comparisons
of taxa within the Geomyidae (Rbp3 and 12S rRNA).
Based on this test, heteromyid taxa (generic or family
level) and Cytb sequences from all taxa appear to be
evolving at neutral rates in all comparisons. In addi¬
tion, the HKAtest (Hudson et al. 1987) indicated that
0 values between Rbp3 and Cytb were significantly
different (P = 0.0016). The HKAtest did not detect any
other significant differences in 24 additional pairwise
comparisons of genera and loci, which suggests that
purifying selection was responsible for a slower rate
of molecular evolution at Rbp3 in Geomys but that the
remaining sequences were evolving at a neutral rate.

Coalescence theory. —The mean rates of evo¬
lution (substitutions per site per million years) were
0.0023, 0.0067, and 0.0138 for Rbp3, 12S rRNA, and
Cytb , respectively. The coefficient of variance for Rbp3
and 12S rRNA were high (0.4847, 0.6783) but low for
Cytb (0.0872). A one-way ANOVA(F = 2.1832 x 1012,
P ~ 0.0000) rejected the null hypothesis of equal rates
among the three datasets, indicating independent rates
of evolution for each gene. In addition, trees obtained
from each of the three genes used in the BEAST analy¬
sis depicted more recent divergence times for species
of Geomys based on Rbp3 than for the other two genes
(Fig. 2).

Discussion

The observation (Chambers et al. 2009) thatRfy?3
sequences obtained from several species of Geomys
were evolving at rates slower than sequences obtained
from other genes for the same taxa was re-examined
using genetic distances (uncorrected-P), relative rate
test (Tajima 1993), neutrality tests (Tajima’s D statis¬
tic, Tajima 1989; HKAtest, Hudson et al. 1987), and
coalescence theory (BEAST, Drummond and Rambaut
2007). All analyses, whether visual (comparison of
genetic distances) or statistically supported (Tajima’s
relative rate test, Tajima’s test of neutrality, HKAtest,
or coalescence theory) indicated that species of Geomys
were evolving at a rate slower compared to members
of the Heteromyidae. Also, other pocket gopher
genera ( Cratogeomys, Orthogeomys, Pappogeomys,
and Thomomys) appeared to evolve more slowly than
their heteromyid counterparts, although low sample
sizes prevented meaningful statistical analyses in some
cases.

Although the various analyses performed in this
study revealed differences in the molecular evolution
of Rbp3 in geomyids and heteromyids, with geomyids
consistently possessing a slower rate of evolution, it
was not clear from a molecular standpoint why geomy¬
ids possessed a slower rate. To further investigate this
phenomenon, we determined the number of variable
sites per codon position (1st, 2nd, and 3rd) for DNA
sequences obtained from the two protein-coding genes
{Rbp3 and Cytb)', the 12S rRNA gene was not included

as it is not a protein-coding locus. The average number
of variable sites (by position) was determined at the
generic and familial levels for geomyids and hetero¬
myids (Table 5). A chi-square test was used to detect
differences in the observed number of variable sites
(represented by the number of changes per position
in Rbp3) versus the expected number of variable sites
(represented by the number of changes per position in
Cytb). Cytb was selected as the “expected” value to
approximate a neutral rate. Significant differences (P
< 0.05) were detected among taxa for Rbp3 relative to
Cytb, with the genera of geomyids possessing a signifi¬
cantly lower number of substitutions, in the 1st and 3rd
positions relative to the other taxa (Table 5).

At least four scenarios are possible for explain¬
ing the low level of genetic variation in the Rbp3 gene
in Geomys and for pocket gophers in general. First,
the product of being fossorial has resulted in pocket
gophers being distributed in small isolated populations,
susceptible to inbreeding, and generally characterized
by low levels of heterozygosity, etc. Also, it is well
known that glacial periods had a major impact on the
distribution and speciation of Geomys (Blair 1954; Rus¬
sell 1968; Penney and Zimmerman 1976; Heaney and
Timm 1983; Mauk et al. 1999) by producing population
bottlenecks during glacial maxima. These events may
have acted to homogenize or constrain evolution of
the Geomys genome. However, these arguments seem
unlikely given that levels of genetic variation reported

Bradley et al.— Rbp3 in Geomyid Rodents

11

Figure 2. Coalescence trees were generated using the BEAST analysis (Drummond and Rambaut 2007) and DNA
sequences obtained from the Rbp3 (top), 12S rRNA (middle), and Cytb (bottom) genes. The GTR + I + G model of
substitution and two combined runs of 10,000,000 generations (with a 10% burn-in) were used for tree construction.
Two fossil calibrations of ancestral taxa (Castorimorpha - 54.4 MYA, McKenna 1960; Geomyoidea - 45.45 MYA,
Walsh 1991) were used as priors on the tree. Numbers at nodes reflect approximate coalescence times.

Table 5. Number of variable nucleotide sites per codon position for the protein-coding genes, interphotoreceptor retinoid binding protein gene fRbpi^) and
mitochondrial cytochrome-b fCyt b), respectively. For each taxonomic group and associated genes, the following is depicted: average number of bases
examined (ANBE), total number of changes per gene, number of changes at the 1st position, number of changes at the 2nd position, number of changes at
the 3rd position, and statistical significance (SIGN). A chi-square test (P < 0.05) was used to determine if the number of nucleotide changes (by position)
in the Rbp3 gene (treated as observed data) was significantly different relative to changes per position in the Cyt b gene (treated as expected data).

Occasional Papers, Museum of Texas Tech University

SIGN

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Yes

Yes

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No

No

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Bradley et al.— Rbp3 in Geomyid Rodents

for 12S rRNAand Cytb (Jolley et al. 2000; Sudman et
al. 2006; Chambers et al. 2009) are similar to genetic
divergences obtained through comparisons with other
species of rodents. It is possible that genetic drift “tar¬
geted” the Rbp3 gene but did not reduce genetic varia¬
tion in 12S rRNA and Cytb ; however, this hypothesis
should be further examined as the results of Tajima’s
neutrality test indicated that population bottleneck and
positive selection were both viable explanations for the
reduction in molecular evolution of Rbp3 in Geomys
and other geomyids.

Second, contemporary species of Geomys may
have diverged recently and, consequently, should
possess low levels of genetic variation at Rbp3. How¬
ever, several lines of data oppose this hypothesis. For
example, fossil evidence (Russell 1968) places the
origin of modern species of Geomys to be at least 5-7
million years ago (MYA). In addition, Jolley et al.
(2000) used rates of molecular divergence estimated
from 12S rRNA sequences to hypothesize that extant
species of Geomys diverged between 2.5 and 5.7 MYA
and a similar value (2.5-7 MYA) is obtained if DNA
sequences from Cytb (Sudman et al. 2006; Chambers
et al. 2009) are used with a molecular divergence rate
of approximately 3% per million years. Although co¬
alescence times obtained herein (Fig. 2) for Cytb (15.75
MYA) are greater than those reported by Sudman et al.
(2006) and Chambers et ah (2009), coalescence times
for Rbp3 and 12S rRNA (8.7 MYA and 7.31 MYA,
respectively) are comparable to fossil estimates and
previous molecular hypotheses. Consequently, a recent
divergence time for Geomys and concomitant reduction
in genetic divergence for Rbp3 seems unlikely.

Third, Rbp3 encodes a large glycolipoprotein
in the interphotoreceptor matrix and is thought to
play a role in retinoid transport between retinal pho¬
toreceptors and pigment epithelial cells (Borst et al.
1989). It is possible, during evolution of the fossorial

13

lifestyle characteristic of Geomys and other species of
pocket gophers, that molecular evolution in Rbp3 was
somehow constrained as a possible consequence of a
reduced emphasis on vision as a result of their fossorial
lifestyle. Similar observations have been reported in
studies of other fossorial genera of mammals, includ¬
ing Ctenomys (Borghi et al. 2002) and Notoryctes
(Springer et ah (1997). However, Feldman and Phil¬
lips (1984) concluded that Geomys possess a similar
retinal pigment epithelium to that observed in other
diurnal species (e.g. tree squirrels, ground squirrels,
and voles) and actually may have limited visual acuity
under low light conditions. We tested this hypothesis
by comparing geomyids to heteromyids, and based on
data presented herein, we cannot reject a connection
between fossoriality and the reduction of molecular
evolution in Rbp3.

Fourth, selective forces may be acting to reduce
or constrain genetic variation at Rbp3. Results from
Tajima’s neutrality test (Tajima 1989), HKAtest (Hud¬
son et ah 1987), and BEAST (Drummond and Rambaut
2007) rejected a neutral model of evolution for Rbp3 in
Geomys and geomyids in some analyses. In addition,
Tajima’s neutrality test and the HKAtest also indicated
that positive selection was a possible explanation for a
slower rate of molecular evolution in Geomys , although
the mechanisms were not clear.

At this time, there are insufficient data to deter¬
mine if the reduction of genetic variability in Rbp3
in geomyid rodents is a product of fossoriality (small
population size, bottlenecks, reduction in development
of the geomyid eye, etc.), population dynamics, age of
the geomyid lineage, or selective forces (positive selec¬
tion). Support for positive selection was identified in
some analyses, although interpretations of these results
were not unambiguous. Further tests of other fosso¬
rial mammals (moles, mole rats, ctenomyids, etc.) are
needed before broader conclusions can be made.

Acknowledgments

We thank the following museums and curators for
providing tissue samples: Natural Science Research
Laboratory at the Museum of Texas Tech University
(R. J. Baker) and Louisiana State University Museum

of Natural Science (M. S. Hafner). We thank S. B.
Ayers, A. P. Clinton, M. S. Corley, R. M. Duplechin,
M. R. Mauldin, N. Ordonez-Garza, and E. Vargas for
commenting on earlier versions of this manuscript.

14

Occasional Papers, Museum of Texas Tech University

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16

Occasional Papers, Museum of Texas Tech University

Addresses of authors:

Robert D. Bradley

Department of Biological Sciences and

Natural Science Research Laboratory, The Museum

Texas Tech University

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robert. bradley@ttu. edu

Cody W. Thompson

Ryan R. Chambers

Oregon State Police Forensic Services Division
Portland Forensic Laboratory
13309 SE 84 th Avenue, Suite 200
Clackamas, OR 97015
ryan. r. chambers@gmail. com

Department of Biological Sciences
Texas Tech University
Lubbock, TX 79409-3131
cody. thompson@ttu. edu

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